PROJECT SUMMARY Tuberculosis (TB) is the leading cause of death from a single infectious disease agent worldwide. Vaccination is the most cost-effective control intervention for any infectious disease. Bacille Calmette?Gurin (BCG) remains the most widely used vaccine in human history, but as currently used it has failed to control TB. Thus, the development of improved vaccines against TB therefore remains a high global priority. Recent studies indicate that BCG, when modified, administered through alternate routes, or used in revaccination, offers improved protection, suggesting that it is well poised to make comeback. We have generated a novel recombinant BCG known as BCG-disA-OE which is engineered to overexpress c-di-AMP, a potent STING agonist Our preliminary studies show that BCG-disA-OE is more effective than BCG-WT (wild type BCG) in prevention of disease (POD) following TB challenge and also as an immunotherapy for non-muscle invasive bladder cancer (NMIBC) where intravesical BCG is currently the first-line therapy. Guinea pigs vaccinated with BCG-disA-OE were significantly better protected against aerosol challenge with virulent M.tb than with BCG-WT, and we found that BCG-disA-OE also showed superior efficacy BCG-WT in rat and mouse models of NMIBC. Compared with BCG-WT, BCG-disA-OE leads to more potent pro-inflammatory cytokine responses in macrophage and bladder cancer cells, a higher degree of proinflammatory epigenetic marks, and greater myeloid cell polarization towards the M1 phenotype?changes that are all consistent with enhanced ?trained immunity?, a newly discovered phenomenon characterized by epigenetic and functional reprogramming of innate immune cells. In this application, our central scientific premise is that the addition of STING agonist overexpression to BCG will augment trained immunity changes in macrophages and provide more effective protection against TB. To test these hypotheses, in Aim 1 we will determine the protective efficacy of BCG-disA-OE versus BCG-WT against TB disease in non-human primates (NHPs) and against reactivation of latent tuberculosis in mice. In Aim 2 we will evaluate trained immunity changes induced by the two BCG strains in macrophage cell lines, as well as primary murine, NHP, and human (leukopacs from healthy donors) macrophages. We will include serial sampling of NHP mononuclear cells from blood and BAL of NHPs as well as their post-mortem tissues obtained under Aim 1. In Aim 3, we will characterize polarization of myeloid and lymphoid cell populations in the TB granuloma induced by prior vaccination with BCG-WT versus BCG-disA-OE in mouse and NHP models of TB.